PROJECT SUMMARY/ABSTRACT There are no effective non-invasive diagnostic imaging approaches to accurately stratify and monitor immunotherapy in adults and children with glioma. PET imaging, utilizing radiolabeled antibody fragments, minibodies (Mb), or diabodies (Db), stably chelated to radiometals, is a promising option for the safe and effective direct quantification of cell surface markers in glioma patients that reflect dynamic immunological processes that bear directly on immunotherapeutic effectiveness. However, development of PET for molecular imaging of markers to guide immunotherapy is in its nascent stage. The long-term goal of this application is to translate effective antibody fragment-based radiotracers for non-invasive diagnostic imaging before and during immunotherapy to meaningfully impact clinical decision making for patients on immunotherapies. The overall objective of this application is to validate radiotracer compositions for unique situations in monitoring immunotherapies in patient-relevant murine glioma models. The rationale for the proposed research is that non- invasive diagnostic imaging with radiotracers quantifying an important immune target, activated T-cells, and immunosuppressive cells to prevent and shorten the duration of ineffective therapies. The central hypothesis is that radiolabeled CD11b, EphA2, and CD69, antibody-based PET tracers can effectively guide immunotherapies for malignant gliomas. In Aim 1, CD11b will be quantified in glioma models by PET with Cu-64-labeled anti- CD11b Mb/Db to quantify immunosuppressive tumor-associated myeloid cells (TAMC) before and during TAMC- targeted immunotherapies. In Aim 2 preclinical PET will be employed to quantify EphA2 expression levels in gliomas. EphA2, a highly relevant clinical immunotherapy target, will serve as a ?proof of principle? antigen, and provide a base to develop comprehensive antigen-PET strategies. Standard uptake values (SUV) of Cu-64- labeled anti-EphA2 Mb/Db will be used to quantify EphA2 levels on glioma cells in syngeneic orthotopic models with a range of EphA2 levels and identify glioma-bearing mice that will respond to immunotherapies. In Aim 3, responses to glioma immunotherapy will be assessed by CD69 PET with Cu-64-labeled anti-CD69 Mb/Db to quantify T-cell activation and predict survival rates of glioma-bearing mice following T-cell-mediated immunotherapies. The use of robust imaging probe chemistry, adult and pediatric murine glioma models, and immunotherapy approaches, will validate novel human/mouse cross-reactive Mb/Db for their translational capacity. If successful, this proposal will radically change the way gliomas are stratified and monitored on immunotherapy trials, using real-time molecular PET imaging to determine which subjects to enroll and when to stop or continue therapy. Outcomes from this research will greatly improve response rates to immunotherapy while reducing unnecessary treatment-related side effects, ineffective and costly treatments, in an era of precision medicine with increased treatment options.